KR100467165B1 - Composition for detecting β-1,3-glucan, preparation method thereof and diagnostic kit detecting β-1,3-glucan - Google Patents
Composition for detecting β-1,3-glucan, preparation method thereof and diagnostic kit detecting β-1,3-glucan Download PDFInfo
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- KR100467165B1 KR100467165B1 KR10-2001-0003036A KR20010003036A KR100467165B1 KR 100467165 B1 KR100467165 B1 KR 100467165B1 KR 20010003036 A KR20010003036 A KR 20010003036A KR 100467165 B1 KR100467165 B1 KR 100467165B1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
본 발명은 시료내에 존재하는 극미량의 β-1,3-글루칸을 검출할 수 있는 조성물 및 그것을 제조하는 방법 및 β-1,3-글루칸 검출용 키트에 관한 것으로, 본 발명의 조성물은 칼슘 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타낸다. 본 발명의 조성물은 체액, 또는 곤충의 플라즈마와 혈구 용혈물의 혼합물인 시료를 컬럼 크로마토그래피로 정제함에 의해 제조할 수 있으며, 구체적으로는 시료 및 분리 공정에 존재하는 칼슘이온을 킬레이팅하기에 충분한 킬레이팅제 존재하에서 곤충의 체액, 또는 플라즈마와 혈구 용혈물의 혼합물인 시료를 얻고, 얻어진 시료를 상기 시료 및 분리 공정에 존재하는 칼슘이온을 킬레이팅하기에 충분한 킬레이팅제를 함유하는 용매 또는 완충액으로 처리하여 분획을 얻고, 얻어진 분획 중 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 분획을 선택하는 것에 의해 제조할 수 있다. 본 발명에 따른 조성물을 이용하여, 검체로부터 시료를 채취하고, 상기 시료에 본 발명에 따른 조성물과 칼슘 이온을 첨가하고, 상기 시료에서의 페놀옥시데이즈 활성을 측정함으로써 β-1,3-글루칸을 검출할 수 있다.The present invention relates to a composition capable of detecting a trace amount of β-1,3-glucan present in a sample, a method for producing the same, and a kit for detecting β-1,3-glucan, wherein the composition of the present invention is in the presence of calcium. The phenol oxides activity is shown by β-1,3-glucan. The compositions of the present invention can be prepared by column chromatography of samples of body fluids, or mixtures of plasma and hemolytic hemolysis of insects, specifically killing enough calcium ions to chelate calcium ions present in the sample and separation process. Obtaining a sample which is a body fluid of an insect, or a mixture of plasma and blood cell hemolysis in the presence of a rating agent, and treating the obtained sample with a solvent or buffer containing sufficient chelating agent to chelate the calcium ions present in the sample and the separation process. To obtain a fraction, and can be prepared by selecting a fraction which exhibits phenol oxidase activity by β-1,3-glucan in the presence of calcium ions in the obtained fraction. Using the composition according to the present invention, a sample is taken from a sample, the composition and calcium ions according to the present invention are added to the sample, and β-1,3-glucan is determined by measuring phenol oxidase activity in the sample. Can be detected.
Description
본 발명은 β-1,3-글루칸 검출용 조성물, 그것의 제조방법 및 β-1,3-글루칸 검출용 키트에 관한 것이다.The present invention relates to a composition for detecting β-1,3-glucan, a preparation method thereof, and a kit for detecting β-1,3-glucan.
암 환자나 장기이식 수술환자, AIDS 환자 등 면역기능이 저하된 환자에게 전신 진균 감염증이나 프로토조아와 같은 원생동물의 감염이 증가되는 사실은 의료계에서 심각한 문제로 대두되고 있으며, 이로 인한 사망률도 점점 증가되고 있다. 이러한 면역기능이 저하된 환자들에게 조기에 진균 감염 여부를 판단하여 적절한 항진균제를 투여하는 것은 매우 필요한 조치이나 현재 조기 진균 감염 여부를 판단하는 것에 어려움이 있다. 또한, 많은 AIDS 환자가 주폐포자충(Pneumocystis carinii)으로 인한 폐렴으로 사망하고 있고 최근 주폐포자충의 세포벽성분으로 β-1,3-글루칸이 존재함이 보고되고 있다 (Kottom et al., J. Biol. Chem. (2000), 275(51), pp. 40628-34).Increasing numbers of protozoan infections, such as systemic fungal infections and protozoa, are becoming a serious problem in the medical community, including cancer, organ transplant patients, and AIDS patients. It is becoming. It is very necessary to determine whether the fungal infection is early and administer appropriate antifungal drugs to patients with reduced immune function, but it is difficult to determine whether the fungal infection is present. In addition, many AIDS patients are dying from pneumonia caused by Pneumocystis carinii, and recently, β-1,3-glucan is reported as a cell wall component of pneumocystis carinii (Kottom et al., J. Biol. Chem. (2000), 275 (51), pp. 40628-34).
환자 진단의 경우 면역기능이 저하된 환자의 진균 감염 여부를 진단하기 위해서는, 지금까지는 진균학적 방법 즉, 환자의 혈액을 채취하여 배양을 통해 진균의 감염 여부를 판단하고 있으나 이 방법은 배양 시간이 2-5일이 요구되어 치료시기를 맞추지 못하는 단점이 있다. 최근에 진균이 가진 항원을 이용하는 방법이나 진균의 대사산물을 이용한 진단 방법이 제시되었으나 후자의 경우 여러 대사산물을 모두 분석해야 할 뿐 아니라 진균 대사산물의 빈번한 변이 유도에 의해 그 감도나 정확도가 낮다는 문제점이 있다. 이러한 이유로 전신 진균 감염의 초기 단계에 감염 환자의 혈액중에 미량 존재하는 β-1,3-글루칸을 정확하게 인식하는 시스템을 찾기 위하여 많은 연구가 진행되었다.In the case of patient diagnosis, in order to diagnose fungal infection of a patient with reduced immune function, up to now, fungal methods, ie, collecting blood from a patient, have been used to determine whether the fungus is infected. -5 days is required, there is a disadvantage that can not be timed treatment. Recently, a method of using an antigen of a fungus or a diagnosis method using a fungal metabolite has been proposed, but in the latter case, it is necessary to analyze all the metabolites, and the sensitivity or accuracy of the fungal metabolite is low due to frequent induction of mutations. There is a problem. For this reason, many studies have been conducted to find a system that accurately recognizes β-1,3-glucan in the blood of infected patients in the early stage of systemic fungal infection.
한편, 가재나 어패류의 인공 양식에서 곰팡이가 양식조에 감염되면 가재나 어패류가 다량 괴사하므로 이로 인한 양식업계의 경제적 피해가 심각하다. 이러한 경우에도 진균의 감염을 초기에 진단할 수 있다면 적절한 조치를 취할 수 있어 수산 양식을 효율을 향상시킬 수 있다.On the other hand, if the fungus is infected with aquaculture tanks in the artificial culture of crawfish and fish and shellfish, the economic damage to the aquaculture industry is serious due to the necrosis of the crawfish and seafood. Even in these cases, if the fungal infection can be diagnosed early, appropriate measures can be taken to improve the efficiency of fish farming.
곤충에서의 멜라닌 형성은 체내에 존재하는 페놀성 물질의 산화로 시작되는데, 이 과정에 작용하는 효소인 페놀옥시데이즈는 곤충의 체내에서 평상시에는 불활성 형태인 프로페놀옥시데이즈 형태로 존재하다가 외부 이물질에 의해 활성화되는 프로페놀옥시데이즈 연쇄 반응의 최종산물의 자극에 의해 최종 활성 형태인 페놀옥시데이즈로 전환되는 것으로 알려져 있다. 이러한 프로페놀옥시데이즈의 활성화는 미생물의 세포벽 성분인 β-1,3-글루칸, 리포폴리사카라이드, 펩티도글리칸 등에 의하여 개시되는 것으로 보고되었다.Melanin formation in insects begins with the oxidation of phenolic substances present in the body. The enzyme phenoloxidase, which acts in this process, is present in the insect body in the form of propphenol oxidase, which is normally in an inactive form. It is known to be converted to the final active form of phenoloxidases by stimulation of the final product of the prophenoloxides chain reaction activated by. The activation of such prophenoloxides has been reported to be initiated by cell wall components of microorganisms β-1,3-glucan, lipopolysaccharide, peptidoglycan and the like.
완전변태 곤충의 생체 내에는 프로페놀옥시데이즈가 존재하며, β-1,3-글루칸 또는 리포폴리사카라이드에 의해 활성화되는 캐스케이드반응에 의해 페놀옥시데이즈로 활성화된다. 일련의 캐스케이드 단계로 이루어지는 이 반응 시스템은 외부에서 침입한 병원균이나 이물질, 혹은 자신의 혈구 세포의 탈과립화반응에 의해 유도되는 내부인자 등에 의하여 쉽게 활성화되어 페놀옥시데이즈로 변화되어 카테콜아민류를 이용하여 멜라닌을 형성해 버린다. 따라서 이 반응 시스템을 생체 외로 분리하기 어려웠다.Prophenoloxides exist in vivo of morphotropic insects and are activated to phenoloxidases by a cascade reaction activated by β-1,3-glucan or lipopolysaccharide. This reaction system, which consists of a series of cascade steps, is easily activated by internal factors induced by externally invading pathogens or foreign substances or degranulation reaction of its own blood cells, and is converted into phenol oxidases to use melanin using catecholamines. Will form. Therefore, it was difficult to separate this reaction system in vitro.
Ashida 등은 미국특허 4,970,152에서 엔도톡신과는 반응하지 않고 펩티도글리칸 및 β-1,3-글루칸과는 반응하는 누에의 플라즈마로부터 얻은 조성물을 이용하여 펩티도글리칸 또는 β-1,3-글루칸을 검출하는 방법을 제시하였다. 이 특허에서는 친화 크로마토그래프에 의해 펩티도글리칸과 반응하는 단백질을 제거함으로써 β-1,3-글루칸을 특이적으로 인식하는 조성물을 제공하였다.Ashida et al., In US Pat. No. 4,970,152, use peptidoglycan or β-1,3-glucan using a composition obtained from a plasma of silkworms that do not react with endotoxin but react with peptidoglycan and β-1,3-glucan. A method of detecting is presented. This patent provides a composition that specifically recognizes β-1,3-glucan by removing proteins that react with peptidoglycans by affinity chromatography.
Ashida 등은 Eur. J. Biochem, 188, 507-515(1990) 모기 유충으로부터 분리한, β-1,3-글루칸을 인식하는 조성물을 제시하면서 프로페놀옥시데이즈의 활성에 2가 이온이 중요한 역활을 한다고 밝혔다.Ashida et al. Eur. J. Biochem, 188, 507-515 (1990) Presenting a composition that recognizes β-1,3-glucan, isolated from mosquito larvae, revealed that divalent ions play an important role in the activity of prophenoloxides.
미국특허 5,266,461에는 리물러스(Limulus) 혈구 혼합물로부터 분리한, β-1,3-글루칸을 인식하는 조성물이 제시되었다. 그러나 리물러스는 희귀동물로 대부 분의 국가에서 자연보호동물로 지정되어 있다는 문제점이 있다.U.S. Patent 5,266,461 discloses a composition that recognizes β-1,3-glucan, isolated from a Limulus blood cell mixture. However, Limulus is a rare animal and has been designated as a conservation animal in most countries.
본 발명은 β-1,3-글루칸 검출용 조성물에 관한 것이다.The present invention relates to a composition for detecting β-1,3-glucan.
본 발명은 β-1,3-글루칸 검출용 조성물의 제조방법에 관한 것이다.The present invention relates to a method for producing a composition for detecting β-1,3-glucan.
본 발명은 β-1,3-글루칸 검출 방법에 관한 것이다.The present invention relates to a β-1,3-glucan detection method.
본 발명은 β-1,3-글루칸 검출용 키트에 관한 것이다.The present invention relates to a kit for detecting β-1,3-glucan.
도 1은 갈색거저리의 체액에서 분리한 페놀옥시데이즈 조성물의, 칼슘 이온 및 β-1,3-글루칸 존재하에서의 반응시간에 따른 페놀옥시데이즈 활성 그래프이다.1 is a graph of phenol oxidase activity according to the reaction time in the presence of calcium ions and β-1,3-glucan of the phenol oxidase composition separated from the body fluid of brown rice bran.
도 2는 갈색거저리의 체액에서 분리한 페놀옥시데이즈 조성물의 β-1,3-글루칸 농도에 따른 페놀옥시데이즈 활성의 표준곡선이다.Figure 2 is a standard curve of phenol oxidase activity according to the β-1,3-glucan concentration of the phenol oxidase composition separated from the body fluid of brown rice bran.
도 3은 갈색거저리의 체액에서 분리한 페놀옥시데이즈 조성물의 리포폴리사카라이드에 대한 검출의 특이성을 조사한 그래프이다.3 is a graph showing the specificity of the detection of lipopolysaccharide of the phenol oxides composition isolated from the body fluid of brown rice wine.
도 4는 갈색거저리의 체액에서 분리한 페놀옥시데이즈 조성물의 펩티도글리칸에 대한 검출의 특이성을 조사한 그래프이다.4 is a graph showing the specificity of the detection of peptidoglycan of the phenol oxides composition isolated from the body fluid of brown rice bran.
도 5는 갈색거저리의 플라즈마, 혈구용혈물, 체액에서 각각 얻은 조성물이 β-1,3-글루칸에 의해 유도되어 페놀옥시데이즈 활성을 나타내는 정도를 비교한 그래프이다.Figure 5 is a graph comparing the degree of phenol oxidase activity was induced by the β-1,3-glucan, respectively, the composition obtained from plasma, blood cell hemoglobin, and body fluid of brown rice wine.
도 6은 갈색거저리의 플라즈마에서 얻은 페놀옥시데이즈 조성물의 β-1,3-글루칸의 농도에 따라 나타나는 페놀옥시데이즈 활성을 관찰한 그래프이다.FIG. 6 is a graph illustrating phenol oxidase activity according to the concentration of β-1,3-glucan of the phenol oxidase composition obtained in the plasma of brown rice wine.
도 7은 한국산 참검정풍뎅이 유충의 플라즈마에서 분리한 페놀옥시데이즈 조성물, 혈구용혈물 및 이들의 혼합물의 β-1,3-글루칸에 대한 페놀옥시데이즈 활성을 비교한 그래프이다.Figure 7 is a graph comparing the phenol oxidase activity against β-1,3-glucan of the phenol oxidase composition, blood cell hemolytes and mixtures thereof isolated from the plasma of Korean black beetle larvae.
도 8은 한국산 참검정풍뎅이 유충의 플라즈마에서 분리한 페놀옥시데이즈 조성물과 혈구용혈물의 혼합물의 칼슘이온 및 β-1,3-글루칸 존재하에서 시간에 따른 페놀옥시데이즈 활성 그래프이다.8 is a graph of phenol oxidase activity with time in the presence of calcium ions and β-1,3-glucan of the mixture of phenol oxidase composition and blood cell hemolysin isolated from plasma of Korean black beetle larvae.
도 9는 암 환자의 혈액을 대상으로, 페놀옥시데이즈 조성물을 이용한 β-1,3-글루칸 검출 결과를 나타내는 그래프이다.9 is a graph showing β-1,3-glucan detection results using phenol oxidase compositions in blood of cancer patients.
도 10은 종양 및 염증성 질환을 가진 환자의 혈액을 대상으로, 페놀옥시데이즈 조성물을 이용한 β-1,3-글루칸 검출 결과를 나타내는 그래프이다.FIG. 10 is a graph showing β-1,3-glucan detection results using phenol oxidase compositions in blood of patients with tumors and inflammatory diseases. FIG.
도 11은 칸디다증 환자를 대상으로, 페놀옥시데이즈 조성물을 이용한 β-1,3-글루칸 검출 결과를 나타내는 그래프이다.FIG. 11 is a graph showing β-1,3-glucan detection results using phenol oxidase compositions in patients with candidiasis. FIG.
본 발명은 시료내의 β-1,3-글루칸을 검출할 수 있는 조성물, 그것을 제조하는 방법 및 그것을 이용한 β-1,3-글루칸 검출용 키트에 관한 것이다.The present invention relates to a composition capable of detecting β-1,3-glucan in a sample, a method for producing the same, and a kit for detecting β-1,3-glucan using the same.
본 발명에서 페놀옥시데이즈 시스템은 곤충 내에 존재하는, β-1,3-글루칸에 의해 페놀옥시데이즈로 활성화되는 시스템을 의미한다.By phenol oxidase system in the present invention is meant a system that is activated by phenol oxidase by β-1,3-glucan, present in the insect.
본 발명에서 페놀옥시데이즈 조성물은 페놀옥시데이즈 시스템 성분의 전부 또는 일부를 포함하며, 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 조성물을 의미한다.The phenol oxidase composition in the present invention means a composition containing all or part of the phenol oxidase system components, and exhibits phenol oxidase activity by β-1,3-glucan in the presence of calcium ions.
본 발명은 칼슘 존재하에서, β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 조성물에 관한 것이다. 본 발명의 조성물은 곤충의 페놀옥시데이즈 시스템 전부 또는 일부를 포함하며, 그 예로 프로페놀옥시데이즈를 포함한다.The present invention relates to a composition exhibiting phenol oxidase activity by β-1,3-glucan in the presence of calcium. The composition of the present invention includes all or part of the insect phenol oxidase system, for example prophenol oxidase.
또한 본 발명은 β-1,3-글루칸을 바람직하게는 20pg/ml까지 검출할 수 있는조성물에 관한 것이다.The present invention also relates to a composition capable of detecting β-1,3-glucan, preferably up to 20 pg / ml.
이와 같이 β-1,3-글루칸을 검출할 수 있는 본 발명의 조성물은 개체에 캔디다와 같은 진균의 감염 여부, 주폐포자충과 같은 프로토조아의 감염 여부를 확인하는데 이용될 수 있다.As described above, the composition of the present invention capable of detecting β-1,3-glucan may be used to determine whether an individual is infected with a fungus such as candida or protozoa such as alveolar worm.
본 발명에서 곤충은 체내에 페놀옥시데이즈 시스템을 가지고 있는 곤충을 의미하며, 완전변태 곤충이 바람직하다. 예로는 가재나 새우와 같은 갑각류, 딱정벌레목(Coleoptesra) 등을 들 수 있으며, 바람직하게는 딱정벌레목으로서 거저리과, 풍뎅이과 등을 사용할 수 있다.Insects in the present invention means insects that have a phenol oxidase system in the body, the morphological insects are preferred. Examples include crustaceans such as lobsters and shrimps, Coleoptesra, etc. Preferably, the beetles can be used as macrophage, chafer.
또한 본 발명은 칼슘 존재하에서, β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 조성물을 제조하는 방법에 관한 것이다. 본 발명의 방법은 플라즈마와 혈구 용혈물의 혼합물을 시료로 사용하고, 분리 공정 중 칼슘 이온의 생리적 작용을 차단함으로써 β-1,3-글루칸에 의해 활성화되는 페놀옥시데이즈 조성물을 분리할 수 있다.The present invention also relates to a method for producing a composition exhibiting phenol oxidase activity with β-1,3-glucan in the presence of calcium. The method of the present invention uses a mixture of plasma and hemolytic hemolysis as a sample, and can isolate the phenoloxides composition activated by β-1,3-glucan by blocking the physiological action of calcium ions during the separation process.
따라서 본 발명의 방법은 곤충의 플라즈마와 혈구 용혈물의 혼합물인 시료를 얻고, 얻어진 시료를 상기 시료 및 분리 공정에 존재하는 칼슘이온을 킬레이팅하기에 충분한 킬레이팅제를 함유하는 용매 또는 완충액으로 처리하여 분획을 얻고, 얻어진 분획 중 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 분획을 선택하는 것으로 이루어진다.Thus, the method of the present invention obtains a sample which is a mixture of plasma and hemolytic hemolysis of an insect, and treats the obtained sample with a solvent or buffer containing sufficient chelating agent to chelate the calcium ions present in the sample and the separation process. The fractions are obtained and the fractions exhibiting phenol oxidase activity by β-1,3-glucan in the presence of calcium ions in the fractions obtained are selected.
본 발명의 방법의 또다른 예는, 곤충의 플라즈마를 상기 플라즈마 및 분리 공정에 존재하는 칼슘이온을 킬레이팅하기에 충분한 킬레이팅제를 함유하는 용매또는 완충액으로 처리하여 분획을 얻고, 얻어진 분획에 혈구 용혈물 또는 부분 정제된 혈구 용혈물을 첨가하고, 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 분획을 선택하는 것으로 이루어진다.Another example of the method of the present invention is to obtain a fraction by treating an insect plasma with a solvent or buffer containing a chelating agent sufficient to chelate the calcium ions present in the plasma and the separation process to obtain fractions, Hemolyte or partially purified blood cell hemolyte is added and a fraction is selected that exhibits phenoloxidase activity by β-1,3-glucan in the presence of calcium ions.
본 발명의 방법에서 필요한 경우, 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 분획에 혈구 용혈물을 추가로 첨가할 수 있다.If necessary in the method of the present invention, hemoglobin hemolytes may be further added to the fraction showing phenol oxidase activity by β-1,3-glucan in the presence of calcium ions.
본 발명에서는 곤충에 존재하는 페놀옥시데이즈 시스템이 β-1,3-글루칸에 의해 활성화될 뿐 아니라, 칼슘 이온에 의해서도 활성화된다는 사실을 밝혔다. 따라서 곤충으로부터 페놀옥시데이즈 시스템을 분리하기 위해서는 β-1,3-글루칸 뿐 아니라 칼슘 이온에 의한 활성화를 억제하는 것이 필요하다. 이를 바탕으로 본 발명은 곤충 체액 또는 플라즈마와 혈구용혈물로부터, 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 조성물을 제조하는 방법을 제공한다.In the present invention, the phenol oxidase system present in insects was found to be activated not only by β-1,3-glucan but also by calcium ions. Therefore, in order to isolate the phenol oxidase system from insects, it is necessary to inhibit activation by calcium ions as well as β-1,3-glucan. Based on this, the present invention provides a method for preparing a composition exhibiting phenol oxidase activity by β-1,3-glucan in the presence of calcium ions from insect body fluids or plasma and blood cells.
본 발명의 방법은 시료 및 분리 공정에 존재하는 칼슘이온을 킬레이팅하기에 충분한 킬레이팅제 존재하에서 곤충으로부터 체액 또는 플라즈마와 혈구 용혈물인 시료를 얻고, 얻어진 시료를 상기 시료 및 분리 공정에 존재하는 칼슘이온을 킬레이팅하기에 충분한 킬레이팅제를 함유하는 용매 또는 완충액로 처리하여 분획을 얻고, 얻어진 분획 중 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 분획을 선택하는 것으로 이루어진다.The method of the present invention obtains a sample which is a bodily fluid or plasma and a blood cell hemolyte from an insect in the presence of a chelating agent sufficient to chelate the calcium ion present in the sample and separation process, and the obtained sample is obtained from the calcium present in the sample and separation process. Treatment with a solvent or buffer containing sufficient chelating agent to chelate the ions to obtain a fraction, wherein the fraction obtained exhibits phenoloxidase activity by β-1,3-glucan in the presence of calcium ions; Is done.
본 발명의 방법에서, 바람직하게는 곤충으로부터 시료를 채취할 때, 체액 응고를 억제할 수 있는 항응고 완충액을 사용한다. 항응고 완충액은 곤충의 체액 응고를 억제할 수 있는 완충액이면 모두 사용할 수 있으며, 특히 사이트레이트 완충액이 바람직하다.In the method of the present invention, an anticoagulant buffer is preferably used that can inhibit body fluid coagulation when taking samples from insects. The anticoagulant buffer can be used as long as it is a buffer solution capable of inhibiting body fluid coagulation of insects, and citrate buffer is particularly preferable.
또한 본 발명자들은 곤충의 플라즈마와 혈구 용혈물의 혼합물을 시료로 사용함으로써, 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 특이적으로 페놀옥시데이즈 활성을 나타내는 조성물을 얻을 수 있음을 밝혔다. 본 발명의 조성물은 플라즈마와 혈구 용혈물의 혼합물로부터 β-1,3-글루칸을 20pg/ml까지 검출할 수 있다. 플라즈마와 혈구 용혈물의 혼합물은 곤충의 체액을 취한 후 플라즈마와 혈구를 분리하고 분리된 혈구를 용혈시켜 플라즈마에 혼합하거나, 혈구 용혈물 또는 부분 정제된 형구 용혈물을 부분 정제된 플라즈마에 첨가하여 얻을 수 있다. 또 다른 방법으로, 체액 내의 혈구를 분리하지 않고 그대로 일부 또는 전부의 혈구를 용혈시켜 얻을 수 있다. 예를 들어 체액 또는 분리한 혈구를 초음파분쇄 (sonication)하거나 고속원심분리함에 의해 혈구를 용혈시킬 수 있다.The present inventors also found that by using a mixture of insect plasma and hemolytic hemolyte as a sample, a composition exhibiting phenol oxidase activity specifically by β-1,3-glucan in the presence of calcium ions can be obtained. The composition of the present invention can detect β-1,3-glucan up to 20 pg / ml from a mixture of plasma and hemolytic hemolysis. The mixture of plasma and hemolytic hemolytes can be obtained by taking the body fluid of an insect and then separating the plasma and blood cells and bleeding the isolated blood cells into the plasma, or adding blood cell hemolyses or partially purified hemoglobin hemolytes to the partially purified plasma. have. Alternatively, some or all of the blood cells may be hemolyzed as is without separating blood cells in the body fluid. For example, blood cells may be hemolyzed by sonication or high-speed centrifugation of bodily fluids or separated blood cells.
본 발명의 방법에서 시료를 킬레이팅제를 함유하는 용매 또는 완충액로 처리하여 분획을 얻는 공정은 예를 들어 컬럼 크로마토그래피에 의하여 수행될 수 있다.In the method of the present invention, the process of obtaining a fraction by treating the sample with a solvent or buffer containing a chelating agent may be performed by, for example, column chromatography.
본 발명의 방법에서 시료 채취 및 분리 공정에 존재하는 칼슘이온을 킬레이팅하기에 충분한 킬레이팅제로써, 종래에 알려져 있는 킬레이팅제는 특별한 한정없이 사용할 수 있으며, 예를 들어 EDTA, EGTA, 사이트르산 등을 사용할 수 있다. 킬레이팅제의 양은 대상 곤충 시료나 컬럼의 종류, 사용 용매 등 분리 공정 조건에 따라 달라질 수 있으며, 곤충 시료 및 분리 공정에 존재하는 칼슘이온을 킬레이팅하기에 충분한 양이면 된다. 따라서 이 분야의 통상의 전문가들은 과도한 실험 없이 킬레이팅제의 양을 결정할 수 있을 것이다.As a chelating agent sufficient for chelating calcium ions present in the sampling and separation process in the method of the present invention, conventionally known chelating agents can be used without particular limitation, for example, EDTA, EGTA, citric acid Etc. can be used. The amount of the chelating agent may vary depending on the separation process conditions, such as the target insect sample, the type of column, and the solvent used, and may be sufficient to chelate calcium ions present in the insect sample and the separation process. Thus, ordinary experts in the art will be able to determine the amount of chelating agent without undue experimentation.
본 발명에 따른 제조방법에서 사용할 수 있는 용매 또는 완충액의 종류는 특별히 한정되지 않으나, pH가 6.5 이하인 것이 바람직하다. pH가 6.5보다 높은 경우 페놀옥시데이즈 캐스캐이드 반응의 한 성분인 세린 프로테이즈가 활성화되어 프로페놀옥시데이즈를 페놀옥시데이즈로 활성화시켜버리므로 본 발명에 따른 조성물을 얻기 어렵다.The type of solvent or buffer that can be used in the production method according to the present invention is not particularly limited, but the pH is preferably 6.5 or less. When the pH is higher than 6.5, serine protease, which is a component of the phenol oxidase cascade reaction, is activated to activate the prophenol oxidase with phenol oxidase, thus making it difficult to obtain a composition according to the present invention.
본 발명에서 곤충 시료를 킬레이팅제를 함유하는 용매 또는 완충액로 처리하는 방법의 한 예는 컬럼 크로마토그래피로, 레진을 충진시킨 컬럼에 곤충 시료를 로딩한 후 킬레이팅제를 함유하는 용매 또는 완충액로 용출시켜 분획을 얻을 수 있다. 본 발명에서는 친화크로마토그래피와 같은 별도의 까다로운 정제과정없이 컬럼 크로마토그래피를 수행함에 의해 β-1,3-글루칸을 20pg/ml까지 특이적으로 검출할 수 조성물을 정제할 수 있다.In the present invention, an example of a method of treating an insect sample with a solvent or a buffer containing a chelating agent is column chromatography. The insect sample is loaded into a resin-filled column, followed by a solvent or a buffer containing a chelating agent. Elution can give a fraction. In the present invention, by performing column chromatography without a separate difficult purification process such as affinity chromatography, the composition capable of specifically detecting β-1,3-glucan up to 20 pg / ml can be purified.
본 발명에서 컬럼 크로마토그래피에 사용할 수 있는 레진은 덱스트란을 원료로 하는 레진이나 비닐을 원료로 하는 레진이 바람직하다. 한 예로 세파덱스 또는 토요펄(Toyopearl)을 사용할 수 있다.In the present invention, the resin that can be used for column chromatography is preferably a resin made of dextran or a resin made of vinyl. For example, Sephadex or Toyopearl can be used.
본 발명에 따른 조성물은 β-1,3-글루칸을 특이적으로 검출할 수 있으므로 β-1,3-글루칸을 세포벽 성분으로 가지는 미생물 감염의 진단에 유용하다.Since the composition according to the present invention can specifically detect β-1,3-glucan, it is useful for the diagnosis of microbial infection having β-1,3-glucan as a cell wall component.
따라서 본 발명은 검체에 β-1,3-글루칸을 세포벽 성분으로 가지는 미생물 감염을 진단하는 방법에 관한 것으로, 본 발명의 방법은 검체로부터 시료를 채취하고, 상기 시료에 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 조성물과 칼슘 이온을 첨가하고, 상기 시료에서의 페놀옥시데이즈 활성을 측정하는 것으로 이루어진다. 한 예로, 본 발명에 따른 방법에서 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 조성물은 시료 및 분리 공정에 존재하는 칼슘이온을 킬레이팅하기에 충분한 킬레이팅제 존재하에서 준비된 곤충의 플라즈마와 혈구 용혈물인 시료를 상기 시료 및 분리 공정에 존재하는 칼슘이온을 킬레이팅하기에 충분한 킬레이팅제를 포함하는 용매로 처리하여 얻어진 분획 중 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈를 활성화시키는 분획을 선택하여 제조되는 조성물일 수 있다.Accordingly, the present invention relates to a method for diagnosing a microbial infection having β-1,3-glucan as a cell wall component in a sample, and the method of the present invention takes a sample from the sample and β-1 in the presence of calcium ions in the sample. It consists of adding the composition and calcium ion which show phenol oxidase activity with, 3-glucan, and measure the phenol oxidase activity in the said sample. In one example, a composition exhibiting phenoloxidase activity by β-1,3-glucan in the presence of calcium ions in the process according to the invention is in the presence of a chelating agent sufficient to chelate the calcium ions present in the sample and separation process. Plasma and blood hemolytes of the prepared insects were treated with a solvent containing a chelating agent sufficient to chelate the calcium ions present in the sample and the separation process in the presence of calcium ions in the fraction of β-1,3-glucan It can be a composition prepared by selecting a fraction for activating phenol oxidase by.
본 발명에 따른 β-1,3-글루칸 검출 방법에서, 검체는 사람을 포함하는 동물이거나 생물이 서식하는 환경일 수 있다. 한 예로, 검체로부터 혈액을 채취하여 진균 감염을 진단할 수 있다. 또 다른 예로, 양식업에서는 양식장의 물을 채취하여 진균과 같이 β-1,3-글루칸을 세포벽 성분으로 가지는 미생물 감염을 진단할 수 있다.In the β-1,3-glucan detection method according to the present invention, the specimen may be an animal including a human or an environment in which an organism lives. For example, blood samples may be taken from a sample to diagnose fungal infections. In another example, aquaculture can collect water from aquaculture farms and diagnose microbial infections with β-1,3-glucan as cell wall components, such as fungi.
β-1,3-글루칸을 세포벽 성분으로 가지는 미생물 감염 진단의 특이성을 향상시키기 위해, 필요한 경우 검체 시료에 존재할 수 있는 리포폴리사카라이드를 제거하는 전처리를 할 수 있다. 예를 들어, 검체 시료를 폴리믹신(polymyxin)과 같이 리포폴리사카라이드와 특이적으로 결합하거나 리포폴리사카라이드를 침전시킬 수 있는 물질로 처리함에 의해 리포폴리사카라이드의 영향을 제거할 수 있다.In order to improve the specificity of diagnosis of microbial infection having β-1,3-glucan as a cell wall component, pretreatment may be performed to remove lipopolysaccharides that may be present in the sample sample if necessary. For example, the effects of lipopolysaccharide can be removed by treating the sample sample with a substance that specifically binds to lipopolysaccharide, such as polymyxin, or can precipitate lipopolysaccharide.
본 발명에서 진균 감염의 진단 방법에 이용될 수 있는 페놀옥시데이즈 활성측정 방법은, 기존에 알려진 페놀옥시데이즈 측정 방법을 그대로 또는 변형시켜 이용할 수 있다. 한 예로 아래에 설명한 4-메틸카테콜/4-하이드록시프롤린에틸에스테르(4-MC/4-HP)를 이용한 발색 반응이나 도파민을 이용한 멜라닌 형성 반응을 이용하여 흡광도를 측정함으로써 페놀옥시데이즈 활성을 측정할 수 있으며, 이로부터 쉽게 초기 단계의 진균 감염을 진단할 수 있다.The phenol oxidase activity measuring method which can be used for the diagnosis method of fungal infection in this invention can use the phenol oxidase measurement method known previously, as it is, or may modify it. As an example, phenol oxidase activity was measured by measuring absorbance using a color reaction using 4-methylcatechol / 4-hydroxyprolineethyl ester (4-MC / 4-HP) described below or a melanin-forming reaction using dopamine. It can be measured and from this it is easy to diagnose early stage fungal infections.
또한 본 발명은 β-1,3-글루칸 검출용 키트에 관한 것이다. 본 발명의 β-1,3-글루칸 검출용 키트는 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 조성물을 함유한다. 한 예로, 본 발명에서 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈 활성을 나타내는 조성물은 시료 및 분리 공정에 존재하는 칼슘이온을 킬레이팅하기에 충분한 킬레이팅제 존재하에서 준비된 곤충의 플라즈마와 혈구 용혈물의 혼합물인 시료를 상기 시료 및 분리 공정 상에 존재하는 칼슘이온을 킬레이팅하기에 충분한 킬레이팅제를 포함하는 용매로 처리하여 얻어진 분획 중 칼슘 이온 존재하에서 β-1,3-글루칸에 의해 페놀옥시데이즈를 활성화시키는 분획을 선택하여 제조되는 조성물일 수 있다.The present invention also relates to a kit for detecting β-1,3-glucan. The kit for detecting β-1,3-glucan of the present invention contains a composition exhibiting phenol oxidase activity by β-1,3-glucan in the presence of calcium ions. In one embodiment, a composition exhibiting phenol oxidase activity by β-1,3-glucan in the presence of calcium ions is used for the preparation of insects prepared in the presence of a chelating agent sufficient to chelate calcium ions present in the sample and separation process. A sample, which is a mixture of plasma and blood cell hemolysis, is treated with a solvent comprising a chelating agent sufficient for chelating calcium ions present in the sample and the separation process in the presence of calcium ions in the fraction of β-1,3-glucan It can be a composition prepared by selecting a fraction for activating phenol oxidase by.
본 발명에서 사용하는 완충액 및 페놀옥시데이즈 활성 측정 방법은 다음과 같다.The buffer and phenol oxidase activity measuring method used in the present invention are as follows.
항응고 완충액(pH 5.5): NaCl 15 mM, 트리소디움 사이트레이트 136 mM, 시트르산 26 mM, EDTA 20 mMAnticoagulant buffer (pH 5.5): NaCl 15 mM, trisodium citrate 136 mM, citric acid 26 mM, EDTA 20 mM
β-1,3-글루칸 용액: β-1,3-글루칸(curdlan, Wako Pure Chemical Industries, Ltd.) 10mg을 0.1N NaOH 1ml에 녹인 용액 10 μl과 20mM 트리스 완충액(pH 8.0) 990 μl을 혼합하여 만든 용액β-1,3-glucan solution: 10 μl of 10 mg β-1,3-glucan (curdlan, Wako Pure Chemical Industries, Ltd.) in 1 ml of 0.1 N NaOH and 990 μl of 20 mM Tris buffer (pH 8.0) Solution made by
4-MC/4-HP 발색 반응4-MC / 4-HP color reaction
농도별로 희석한 β-1,3-글루칸 용액 10 μl에 페놀옥시데이즈 조성물 30 μl을 섞어 30 ℃에서 5분간 전반응시키고 20 mM 트리스 완충액(pH 7.5) 437.5 μl와 1M CaCl25μl, 250 mM 4-메틸카테콜(MC) 2μl, 62.5 mM 4-하이드록시프롤린에틸에스테르(HP) 16 μl을 넣어 총량이 500 μl이 되도록 한 후 30℃에서 30분 반응시킨다. 여기에 20% 초산 500 μl을 넣어 반응을 중지시킨 후 520 nm에서 흡광도를 측정한다.Concentration a β-1,3- glucan solution was mixed with 30 μl phenol oxy Days composition in 10 μl at 30 ℃ 5 bungan former reaction and 20 mM Tris buffer (pH 7.5) and 437.5 μl 1M CaCl diluted by 2 5μl, 250 mM 4 -2 μl of methyl catechol (MC) and 16 μl of 62.5 mM 4-hydroxyprolineethyl ester (HP) were added to make the total amount 500 μl, followed by reaction at 30 ° C. for 30 minutes. Add 500 μl of 20% acetic acid to stop the reaction and measure the absorbance at 520 nm.
멜라닌 생성 반응Melanin production reaction
희석한 β-1,3-글루칸 용액 10 μl에 페놀옥시데이즈 조성물 30 μl을 섞어 30 ℃에서 10분간 전반응시킨 후 0.02M 트리스 완충액(pH 8.0) 405 μl, 1M CaCl25μl, 10mM 도파민 용액 50μl을 가하여 30℃에서 60 분 반응시킨다. 400 nm에서 흡광도를 측정하여 표준곡선을 얻는다. 검체에서 채혈한 혈액을 원심분리하여 얻은 혈장을 시료로 사용하여 위와 동일한 방법을 수행하여 400 nm에서 흡광도를 측정한다. 표준곡선으로부터 시료내에 존재하는 β-1,3-글루칸의 양을 결정한다. Mixing the phenol composition oxy Days 30 μl diluted in a β-1,3- glucan solution 10 μl was around 10 minutes of reaction at 30 ℃ 0.02M Tris buffer (pH 8.0) 405 μl, 1M CaCl 2 5μl, 10mM dopamine solution 50μl It was added and reacted at 30 degreeC for 60 minutes. The absorbance is measured at 400 nm to obtain a standard curve. Using the plasma obtained by centrifuging the blood collected from the sample as a sample, the absorbance is measured at 400 nm by the same method as above. From the standard curve, determine the amount of β-1,3-glucan present in the sample.
실시예 1.Example 1.
딱정벌레목 갈색거저리(Tenebrio molitor)의 유충을 얼음 위에서 마취시키고 25G 주사바늘이 연결된 5ml 멸균 주사기에 항응고 완충액을 채워 머리쪽 첫번째 마디에 찔러 유출되는 체액을 마리 당 3방울씩 모았다. 채취한 체액 60ml을 203,006g 속도로 4℃에서 4시간 원심분리한 후 상등액을 필트레이션(0.45um)하여 불순물을 제거하고 55ml을 모았다. 이것을 울트라필트레이션(cut off: 10,000)하여 3ml까지 농축하였다. 토요펄(Toyopearl) HW-55S 레진을 1 x 50 cm 컬럼에 충진시키고 충분한 양의 항응고 완충액으로 컬럼을 세척하였다. 농축된 시료를 로딩하고 항응고 완충액을 0.18ml/분의 속도로 흘려주면서 용출액을 3.8ml씩 모아 280nm에서 흡광도를 측정하여 단백질 농도를 조사하면서 β-1,3-글루칸 용액을 사용한 4-MC/4-HP 발색 반응을 통해, β-1,3-글루칸이 있을 때 발색되는 분획만을 모아 1차 정제된 페놀옥시데이즈 조성물을 3.8 ml을 얻었다. 얻어진 1차 정제된 페놀옥시데이즈 조성물을 사용하여, Ca2+의 존재 또는 부존재 하에서 β-1,3-글루칸 검출 능력을 확인하였다.Larvae of the coleoptera , Tenebrio molitor , were anesthetized on ice and filled with anticoagulant buffer in a 5 ml sterile syringe with a 25G needle and stabbed at the first node on the head to collect 3 drops per fluid. 60 ml of the collected body fluid was centrifuged at 203,006 g at 4 ° C. for 4 hours, and then the supernatant was filtered (0.45 um) to remove impurities and 55 ml were collected. This was ultrafiltered (cut off: 10,000) and concentrated to 3 ml. Toyopearl HW-55S resin was charged to a 1 × 50 cm column and the column was washed with sufficient anticoagulant buffer. Loading the concentrated sample and flowing anticoagulant buffer at a rate of 0.18ml / min, collecting 3.8ml of eluate and measuring the absorbance at 280nm to examine the protein concentration and 4-MC / using β-1,3-glucan solution. Through 4-HP color reaction, only 3.8 ml of the first purified phenol oxides composition was collected by collecting only the fractions developed when β-1,3-glucan was present. Using the obtained primary purified phenol oxidase composition, β-1,3-glucan detection ability was confirmed in the presence or absence of Ca 2+ .
1차 정제된 페놀옥시데이즈 조성물 3.8 ml을 다시 상기와 동일한 컬럼에 로딩하고, 항응고 완충액을 0.16ml/분의 속도로 흘려 주면서 용출액을 3ml씩 모아 280nm에서 흡광도를 측정하여 단백질 농도를 조사하여 β-1,3-글루칸 용액을 사용한 4-MC/4-HP 발색 반응을 통해, β-1,3-글루칸이 있을 때 발색되는 분획만을 모아 2차 정제된 페놀옥시데이즈 조성물을 얻고 이를 최종적으로 페놀옥시데이즈 조성물로 하였다. 최종 Ca2+농도를 각각 0mM과 5mM로 되도록 하고, β-1,3-글루칸 용액 100ng/ml을 함유하는 용액 10ul을 사용하여 페놀옥시데이즈 조성물로 4-MC/4-MP 발색 반응을 수행하고 반응시간에 따라 결과를 확인하였다. 그 결과를 도 1에 나타낸다 (-◆- : 완충액, -■- : 페놀옥시데이즈 조성물, -▲- : 페놀옥시데이즈 조성물+ Ca2+, -×- : 페놀옥시데이즈 조성물 + Ca2++ β-1,3-글루칸). 페놀옥시데이즈 조성물이 β-1,3-글루칸이 존재하는 경우 페놀옥시데이즈 활성을 나타냄을 알 수 있다.3.8 ml of the first purified phenol oxidase composition was loaded on the same column again, and the anticoagulation buffer was flowed at a rate of 0.16 ml / min, and 3 ml of the eluate was collected and the absorbance was measured at 280 nm. 4-MC / 4-HP color reaction using -1,3-glucan solution to collect only the fractions developed when β-1,3-glucan was present to obtain a second purified phenol oxidase composition and finally phenol It was set as the oxidase composition. The final Ca 2+ concentration was brought to 0 mM and 5 mM, respectively, and 4-MC / 4-MP color reaction was carried out with a phenol oxidase composition using 10ul of a solution containing 100ng / ml of β-1,3-glucan solution. The results were confirmed according to the reaction time. The results are shown in Fig. 1 (-◆-: buffer,-■-: phenol oxidase composition,-▲-: phenol oxidase composition + Ca 2+ ,-×-: phenol oxidase composition + Ca 2+ + β -1,3-glucan). It can be seen that the phenol oxidase composition exhibits phenol oxidase activity when β-1,3-glucan is present.
페놀옥시데이즈 조성물의 β-1,3-글루칸의 농도에 따른 페놀옥시데이즈 활성을 측정하고 표준곡선을 정하였다. β-1,3-글루칸의 농도 0 내지 200 pg/ml 에 대해 상기의 발색반응으로 30℃에서 1시간 반응시켜서 520nm에서 흡광도를 측정하였다. 그 결과를 도 2에 나타낸다. 농도와 활성의 상관계수가 0.9862로 나타나 극미량의 β-1,3-글루칸을 검출할 수 있음을 확인할 수 있었다.The phenol oxidase activity according to the concentration of β-1,3-glucan of the phenol oxidase composition was measured and a standard curve was determined. The absorbance was measured at 520 nm by reacting the concentration of β-1,3-glucan at 0 to 200 pg / ml at 30 ° C. for 1 hour. The result is shown in FIG. The correlation coefficient between concentration and activity was 0.9862, indicating that a very small amount of β-1,3-glucan could be detected.
실시예 2.Example 2.
실시예 1의 페놀옥시데이즈 조성물이 β-1,3-글루칸에 대한 특이성이 있는지를 알아보기 위해 리포폴리사카라이드와 펩티도글리칸에 대해 페놀옥시데이즈 활성을 측정하였다. 50mM 트리스 완충액(pH 7.0)에 리포폴리사카라이드(시그마사)와 펩티도글리칸(시그마사)를 각각 현탁시켜 펩티도글리칸은 그대로, 리포폴리사카라이드는 2-3분동안 초음파파쇄한 후 기질로 사용하였다. 리포폴리사카라이드 200pg/ml, 20ng/ml, 20ug/ml의 농도의 기질을 사용하여 최종 Ca2+농도 5mM 및 페놀옥시데이즈 조성물로 4-MC/4-MP 발색 반응을 수행하였으며 β-1,3-글루칸 20ng/ml을 기질로 사용했을 때와 비교하고 그 결과를 도 3에 나타낸다 (□: 페놀옥시데이즈 조성물 + Ca2+를 첨가한 음성대조구 (이하 A), ▨ : A + 리포폴리사카라이드200pg/ml, ▧ : A + 리포폴리사카라이드 20ng/ml, ▤ : A + 리포폴리사카라이드 20ug/ml, ■: A + β-1,3-글루칸 20ng/ml(양성대조구)).In order to determine whether the phenol oxidase composition of Example 1 is specific for β-1,3-glucan, phenol oxidase activity was measured for lipopolysaccharide and peptidoglycan. After lipopolysaccharide (Sigma) and peptidoglycan (Sigma) were suspended in 50 mM Tris buffer (pH 7.0), respectively, the peptidoglycan was intact and lipopolysaccharide was sonicated for 2-3 minutes. Used as substrate. A 4-MC / 4-MP color reaction was carried out with a final Ca 2+ concentration of 5 mM and a phenol oxides composition using a substrate of lipopolysaccharide 200 pg / ml, 20 ng / ml, 20 ug / ml, and β-1, Compared with using 20 ng / ml of 3-glucan as a substrate, the result is shown in Fig. 3 (□: negative control added with phenol oxidase composition + Ca 2+ (hereinafter A), ▨: A + lipopolysaka Ride 200 pg / ml, ▧: A + lipopolysaccharide 20 ng / ml, ▤: A + lipopolysaccharide 20 ug / ml, ■: A + β-1,3-glucan 20 ng / ml (positive control).
또한 펩티도글리칸에 대해서도 상기와 동일한 조건하에서 페놀옥시데이즈 활성을 측정하고 그 결과를 도 4에 나타낸다 (□: 페놀옥시데이즈 조성물+ Ca2+를 첨가한 음성대조구 (이하 A), ▨ : A + 펩티도글리칸 200pg/ml, ▧ : A + 펩티도글리칸 20ng/ml, ▤ : A + 펩티도글리칸 20ug/ml, ■: A + β-1,3-글루칸 20ng/ml (양성대조구)).Peptidoglycan was also measured for phenol oxidase activity under the same conditions as described above, and the results are shown in Fig. 4 (□: negative control with phenol oxidase composition + Ca 2+ added (hereinafter A), ▨: A + Peptidoglycan 200pg / ml, ▧: A + peptidoglycan 20ng / ml, ▤: A + peptidoglycan 20ug / ml, ■: A + β-1,3-glucan 20ng / ml (positive control )).
이와 같이 리포폴리사카라이드 및 펩티도글리칸의 고농도, 즉 20ug/ml에 대해서도, 그 반응시간을 증가시켜도 페놀옥시데이즈 활성을 나타내지 않으므로 본발명의 페놀옥시데이즈 조성물은 β-1,3-글루칸을 특이적으로 검출할 수 있음을 알 수 있다.As such, even with a high concentration of lipopolysaccharide and peptidoglycan, that is, 20 ug / ml, even if the reaction time is increased, phenol oxidase activity is not exhibited. Thus, the phenol oxidase composition of the present invention is β-1,3-glucan. It can be seen that the specific detection.
비교예 1.Comparative Example 1.
딱정벌레목 갈색거저리(Tenebrio molitor)의 유충을 얼음 위에서 마취시키고 25G 주사바늘이 연결된 5ml 멸균 주사기에 항응고 완충액을 채워 머리쪽 첫번째 마디에 찔러 유출되는 체액을 마리 당 3방울씩 모았다. 채취한 체액 85ml을 372 g로 4℃에서 5분간 원심분리한 후 상등액을 플라즈마로, 침전물을 혈구세포로 얻었다. 플라즈마 60ml을 203,006 g의 속도로 4℃에서 4시간 원심분리한 후 상등액을 필트레이션(0.45 um)하여 불순물을 제거하여 58ml을 모으고, 울트라필트레이션(cut off: 10,000)하여 3ml까지 농축하였다. 토요펄 HW-55S 레진을 1 x 50 cm 컬럼에 충진 시키고 충분한 양의 항응고 완충액으로 컬럼을 세척하였다. 농축된 시료를 로딩하고 항응고 완충액을 0.18ml/분의 속도로 흘려 주면서 용출액을 3.8ml씩 모아 280nm에서 흡광도를 측정하여 단백질 농도를 조사하면서 β-1,3-글루칸 용액을 사용한 4-MC/4-HP 발색 반응을 통해, β-1,3-글루칸이 있을 때 발색되는 분획만을 모아 1차 정제된 플라즈마 유래의 페놀옥시데이즈 조성물을 3.8 ml을 얻었다.Larvae of the coleoptera , Tenebrio molitor , were anesthetized on ice and filled with anticoagulant buffer in a 5 ml sterile syringe with a 25G needle and stabbed at the first node on the head to collect 3 drops per fluid. 85 ml of the collected body fluid was centrifuged at 372 g at 4 ° C. for 5 minutes to obtain supernatant as plasma and precipitate as blood cells. 60 ml of plasma was centrifuged at 4 ° C. at 203,006 g for 4 hours, and then the supernatant was filtered (0.45 um) to remove impurities, and 58 ml were collected, and ultra filtration (cut off: 10,000) was concentrated to 3 ml. Toyopearl HW-55S resin was charged to a 1 × 50 cm column and the column was washed with sufficient anticoagulant buffer. Loading the concentrated sample and flowing anticoagulant buffer at a rate of 0.18ml / min, collecting 3.8ml of eluate and measuring the absorbance at 280nm to examine the protein concentration and 4-MC / using β-1,3-glucan solution. Through 4-HP color reaction, only the fractions developed when β-1,3-glucan was collected to obtain 3.8 ml of the phenol oxides composition derived from the first purified plasma.
한편, 침전물인 혈구세포를 취하고 항응고 완충액으로 세척하여 원심분리하여 플라즈마 부분을 제거하였다. 이를 3회 정도 반복하고 혈구세포를 완전히 파괴하기 위해서 초음파분쇄를 실시한 후 372 g로 4℃에서 5분간 원심분리하여 상등액 3ml을 혈구세포 용혈물로 얻었다.Meanwhile, the precipitated blood cells were removed, washed with anticoagulant buffer, and centrifuged to remove the plasma portion. This was repeated about 3 times, and then subjected to ultrasonic grinding to completely destroy the blood cells, and centrifuged at 372 g for 5 minutes at 4 ° C. to obtain 3 ml of the supernatant as hemocytic hemolytes.
부분정제된 플라즈마의 페놀옥시데이즈 조성물, 혈구세포 용혈물 및 실시예 1의 체액으로부터 1차 정제된 페놀옥시데이즈 조성물의 동일 단백질 함량(400ug)에 대해 Ca2+의 존재하에서 각각의 β-1,3-글루칸(농도 2ng/ml) 검출 능력을 비교하고 그 결과를 도 5에 나타내었다. 그림에서 같이 체액으로부터 얻은 페놀옥시데이즈 조성물이 β-1,3-글루칸에 대해 검출능이 있음을 알 수 있다.Each β-1, in the presence of Ca 2+ , for the same protein content (400 ug) of the phenol oxidase composition of the partially purified plasma, the hemocytic cell hemolyte and the phenol oxidase composition first purified from the body fluid of Example 1, The 3-glucan (concentration 2ng / ml) detection capability was compared and the results are shown in FIG. As shown in the figure, it can be seen that the phenol oxidase composition obtained from the body fluid is detectable against β-1,3-glucan.
반면, 상기의 플라즈마유래의 페놀옥시데이즈 조성물를 사용하여 β-1,3-글루칸의 농도에 따른 β-1,3-글루칸 검출능력을 확인하였다. 도 6에서와 같이 플라즈마만에서 얻은 페놀옥시데이즈 조성물에 대해서는 β-1,3-글루칸과 페놀옥시데이즈 활성사이에 상관관계가 없음을 알 수 있다.On the other hand, using the plasma-derived phenol oxides composition was confirmed the β-1,3-glucan detection ability according to the concentration of β-1,3-glucan. As shown in FIG. 6, it can be seen that there is no correlation between β-1,3-glucan and phenol oxidase activity for the phenol oxidase composition obtained by plasma only.
실시예 3.Example 3.
페놀옥시데이즈 조성물이 β-1,3-글루칸을 특이적으로 검출하는지 알아 보기 위하여, 다양한 당을 시료로 사용하여, 위에 기재된 방법에 따라 멜라닌 생성 반응을 수행하였다. 400 nm에서 흡광도를 측정하여 생성된 멜라닌 양을 측정한 결과, 페놀옥시데이즈 조성물은 β-1,3-글루칸 존재하에서만 유의성 있게 멜라닌을 생성하였다. 그 결과를 표 1에 나타낸다.To determine if the phenoloxidase composition specifically detects β-1,3-glucan, a melanogenesis reaction was performed according to the method described above using various sugars as samples. As a result of measuring the amount of melanin produced by measuring absorbance at 400 nm, the phenol oxides composition produced melanin significantly only in the presence of β-1,3-glucan. The results are shown in Table 1.
실시예 4.Example 4.
한국산 참검정풍뎅이(Holotrichia diomphalia) 유충에서 분리한 체액 50ml을 420 g 속도로 4℃에서 20분간 원심분리하여 상등액과 침전물을 각각 플라즈마와 혈구세포로 얻었다. 먼저 침전된 혈구세포 5ml에 50mM 트리스 완충액/1mM EDTA (pH 6.5) 5ml를 가하여 초음파분쇄를 실시한 후 22,000 g 속도로 4℃에서 20분간 원심 분리시켜 상등액을 혈구세포 용혈물로 사용한다.50 ml of the bodily fluid isolated from Korean locust beetle ( Horlotrichia diomphalia ) larvae was centrifuged at 420 g for 20 minutes at 4 ° C to obtain supernatant and precipitate as plasma and blood cells. First, 5 ml of 50 mM Tris buffer / 1 mM EDTA (pH 6.5) is added to 5 ml of precipitated blood cells, followed by ultrasonic disruption, followed by centrifugation at 4 ° C. for 20 minutes at 22,000 g to use the supernatant as hemocytic hemolytes.
한편, 상등액의 플라즈마 50ml을 203,006 g 속도로 4℃에서 4시간 원심분리한 후 상등액을 울트라 필트레이션(cut off: 10,000)하여 3ml까지 농축하였다. 토요펄 HW 55S 레진을 1 X 50 cm 컬럼에 충진 시키고 50mM 트리스 완충액/20mM EDTA (pH 6.5)로 평형화시킨 후 농축된 플라즈마 3ml을 로딩하였다. 50mM 트리스 완충액/20mM EDTA (pH 6.5)를 0.16 mL/분의 속도로 흘리면서, 용출액을 약 3.2mL씩 모아, 280nm에서 흡광도를 측정하여 단백질 농도를 조사하고, 4-MC/4-HP 발색 반응을 통해 5mM Ca2+이 있을 때 발색되는 분획들을 각각 모았다. 이 분획들에 혈구용혈물과 β-1,3-글루칸의 존재하에서 페놀옥시데이즈 활성을 나타내는 분획을 모아 부분정제된 플라즈마액 (3.2ml)으로 하였다.Meanwhile, 50 ml of the supernatant plasma was centrifuged at 4 ° C. for 4 hours at a rate of 203,006 g, and then the supernatant was concentrated to 3 ml by ultra filtration (10,000 off). Toyopearl HW 55S resin was charged to a 1 × 50 cm column, equilibrated with 50 mM Tris buffer / 20 mM EDTA (pH 6.5) and then loaded with 3 ml of concentrated plasma. While flowing 50 mM Tris buffer / 20 mM EDTA (pH 6.5) at a rate of 0.16 mL / min, the eluates were collected at about 3.2 mL, the absorbance was measured at 280 nm, and the protein concentration was examined. The 4-MC / 4-HP color reaction was carried out. The fractions developed when there was 5 mM Ca 2+ were collected. Fractions showing phenol oxidase activity in the presence of hemoglobin blood and β-1,3-glucan were collected in these fractions to obtain a partially purified plasma solution (3.2 ml).
부분정제된 플라즈마액 (단백질함량 : 25 ug), 상기의 혈구용혈물 (단백질 함량 175ug) 및 이들의 혼합물인 페놀옥시데이즈 조성물 (단백질함량비 25ug : 175ug) 각각에 대한 β-1,3-글루칸에 대한 검출능을 측정하고 도 7에 나타내었다. 반응조건은 최종 Ca2+농도를 5mM로 하고, β-1,3-글루칸 농도 0.1 ug/ml을 함유하는 용액 10ul를 사용하여 30분동안 4-MC/4-HP 발색 반응을 수행하였다. 부분정제된 플라즈마액과 혈구용혈물의 혼합물이 β-1,3-글루칸이 있을 경우 페놀옥시데이즈 활성을 나타냄을 알 수 있다.Β-1,3-glucan to each of the partially purified plasma solution (protein content: 25 ug), the hemoglobin hemoglobin (protein content 175 ug), and a mixture of these phenol oxides compositions (protein content ratio 25 ug: 175 ug) The detectability against was measured and shown in FIG. 7. Reaction conditions were the final Ca 2+ concentration of 5mM, 4-MC / 4-HP color reaction was carried out for 30 minutes using 10ul of a solution containing 0.1 ug / ml concentration of β-1,3-glucan. It can be seen that the mixture of the partially purified plasma solution and the blood cell hemolysin shows phenol oxidase activity when β-1,3-glucan is present.
또한, 상기의 페놀옥시데이즈 조성물 사용하여 상기와 동일한 조건에서 반응시간에 따른 β-1,3-글루칸의 검출능을 확인하고 도 8에 나타내었다 (―: 최종 칼슘 농도 5 mM + β-1,3-글루칸 2 μg/ml, □―□: 최종 칼슘 농도 5 mM,―: 20 mM 트리스 완충액, ▲―▲: β-1,3-글루칸 2 μg/ml). 칼슘 이온 및 칼슘 이온존재하에서의 β-1,3-글루칸에 의해 페놀옥시데이즈 활성이 관찰되었다.In addition, using the above phenol oxidase composition was confirmed the detection ability of β-1,3-glucan according to the reaction time under the same conditions as shown above and shown in Figure 8 ( ― : Final calcium concentration 5 mM + β-1,3-glucan 2 μg / ml, □ ― □: final calcium concentration 5 mM, ― : 20 mM Tris buffer, ▲-▲: β-1,3-glucan 2 μg / ml). Phenoloxidase activity was observed by β-1,3-glucan in the presence of calcium ions and calcium ions.
실시예 5.Example 5.
건강한 성인 남녀 11명과 입원 중인 암환자 50명으로부터 채혈하였다. 혈액에 헤파린을 처리한 후 원심분리하여 얻은 혈장을 β-1,3-글루칸 검출용 시료로 사용하였다.Blood was collected from eleven healthy men and women and 50 cancer patients. Plasma obtained by centrifugation after heparin treatment of blood was used as a sample for β-1,3-glucan detection.
환자로부터 얻은 혈장 10μl에 실시예 1의 1차 정제된 페놀옥시데이즈 조성물 10μl을 섞어 4-MC/4-HP 발색 반응을 수행하였다. 520nm에서 흡광도를 측정하고, 표준곡선으로부터 시료에 존재하는 β-1,3-글루칸의 양을 구하였다. 건강한 성인 남녀(11명)로부터 채혈한 혈장에서는 β-1,3-글루칸이 거의 검출되지 않았다. 항암치료로 인하여 면역성이 감소된 고형종양 (solid tumor)과 혈액종양 (hematogenic tumor)을 가진 환자들은 염증성 질환 환자들과 비교하여 현저히 높은 β-1,3-글루칸 농도를 나타내었다(도 9, 괄호안의 숫자는 실행한 실험의 수를 나타내며 β-1,3-글루칸의 농도는 ug/혈청 ml로 나타내고 평균치±SEM임). 도 9에서 기타로 표시된 환자들은 염증성 질환만 가지고 종양은 없는 환자들이다. 도 10에 나타난 바와 같이 염증성 질환 환자들의 경우 β-1,3-글루칸이 거의 검출되지 않았고, 종양과 염증성 질환을 동시에 가지고 있는 환자의 경우 그 값이 더욱 높게 나타났다 (괄호안의 숫자는 실행한 실험의 수를 나타내며 β-1,3-글루칸의 농도는 평균치±SEM임). 후자의 경우는 종양만 있는 환자에 비해 종양과 염증성 질환을 동시에 가지고 있는 환자의 면역성이 더욱 떨어져 진균 감염 정도가 높은 탓으로 추측된다.10 μl of the plasma obtained from the patient was mixed with 10 μl of the first purified phenol oxidase composition of Example 1 to carry out a 4-MC / 4-HP color reaction. The absorbance was measured at 520 nm, and the amount of β-1,3-glucan present in the sample was determined from the standard curve. Almost no β-1,3-glucan was detected in plasma collected from healthy adult men and women (11 patients). Patients with solid tumors and hematogenic tumors with reduced immunity due to chemotherapy showed significantly higher β-1,3-glucan concentrations compared to patients with inflammatory diseases (Fig. 9, parentheses). The numbers inside indicate the number of experiments performed and the concentration of β-1,3-glucan is expressed in ug / ml of serum and is mean ± SEM. In FIG. 9, patients indicated as other are patients with only inflammatory disease and no tumor. As shown in FIG. 10, β-1,3-glucan was hardly detected in patients with inflammatory diseases, and higher in patients with both tumors and inflammatory diseases (the numbers in parentheses indicate the number of experiments performed). Number and the concentration of β-1,3-glucan is the mean ± SEM. The latter case is thought to be due to a higher degree of fungal infection, due to the lower immunity of patients with tumors and inflammatory diseases than patients with tumors alone.
칸디다증(Candidiasis)이 있는 것으로 확진된 환자의 경우 β-1,3-글루칸 농도는 더욱 높았고 칸디다증 환자 중 항진균 치료를 받고 있는 환자들에서는 유의하게 β-1,3-글루칸 농도가 낮았다(도 11, 괄호안의 숫자는 실행한 실험의 수를 나타내며 β-1,3-글루칸의 농도는 평균치±SEM임). 본 발명의 조성물을 사용하여 시료 내의 β-1,3-글루칸 농도를 측정함으로써 유효성 있게 진균 감염 여부를 진단할 수 있음을 알 수 있다.The β-1,3-glucan concentration was higher in patients confirmed to have Candidiasis and significantly lower in β-1,3-glucan concentration in patients receiving antifungal treatment among candidiasis patients (FIG. 11, The numbers in parentheses indicate the number of experiments performed and the concentration of β-1,3-glucan is the mean ± SEM. It can be seen that the fungal infection can be effectively diagnosed by measuring the concentration of β-1,3-glucan in the sample using the composition of the present invention.
본 발명에 의해 β-1,3-글루칸을 세포벽 성분으로 가지는 미생물 감염 여부를 초기에 진단할 수 있다. 면역기능이 저하된 환자들에게 조기에 β-1,3-글루칸을 세포벽 성분으로 가지는 미생물 감염 여부를 판단하여 적절한 항균제를 투여함에 의해 미생물 감염으로 인한 사망을 줄일 수 있으며, 수산 양식업 등에서 곰팡이 감염에 대해 조기에 조치를 취할 수 있어 피해를 감소시킬 수 있다.According to the present invention, it is possible to initially diagnose whether a microorganism is infected with β-1,3-glucan as a cell wall component. By early detection of microbial infections with β-1,3-glucan as a cell wall component to patients with reduced immune function, death of microbial infections can be reduced by administering appropriate antimicrobial agents. Early action can be taken to reduce damage.
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| JPH07184690A (en) * | 1993-11-18 | 1995-07-25 | Wako Pure Chem Ind Ltd | Method for measuring activity of prophenol oxidase activating enzyme and its application |
| JPH11178599A (en) * | 1997-12-22 | 1999-07-06 | Wako Pure Chem Ind Ltd | Reagent for measuring endotoxin and/or peptide glucan |
| KR100264366B1 (en) * | 1995-07-31 | 2000-08-16 | 다나까 모또아끼 | Process for detecting microorganisns |
-
2001
- 2001-01-19 KR KR10-2001-0003036A patent/KR100467165B1/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4970152A (en) * | 1986-12-03 | 1990-11-13 | Wako Pure Chemical Industries, Ltd. | Reagents for determining peptidoglycan and β-1,3-glucan |
| US5266461A (en) * | 1990-06-21 | 1993-11-30 | Seikagaku Corporation | Method for determining (1-3)-β-D-glucan |
| JPH07184690A (en) * | 1993-11-18 | 1995-07-25 | Wako Pure Chem Ind Ltd | Method for measuring activity of prophenol oxidase activating enzyme and its application |
| KR100264366B1 (en) * | 1995-07-31 | 2000-08-16 | 다나까 모또아끼 | Process for detecting microorganisns |
| JPH11178599A (en) * | 1997-12-22 | 1999-07-06 | Wako Pure Chem Ind Ltd | Reagent for measuring endotoxin and/or peptide glucan |
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| KR20010076356A (en) | 2001-08-11 |
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